Synthesis of 5-azacytidine

ABSTRACT

The present invention provides a method for the preparation of 5-azacytidine, wherein 5-azacytidine is represented by the structure: 
     
       
         
         
             
             
         
       
     
     The method involves the silylation of 5-azacytosine, followed by the coupling of silylated 5-azacytosine to a protected β-D-ribofuranose derivative. The coupling reaction is catalyzed by trimethylsilyl trifluoromethanesulfonate (TMS-Triflate).

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/381,275, filed May 2, 2006 now abandoned, entitled “Synthesis of5-Azacytidine,” which is a continuation of U.S. application Ser. No.10/390,526, filed Mar. 17, 2003, entitled “Synthesis of 5-Azacytidine,”now U.S. Pat. No. 7,038,038, issued on May 2, 2006. The foregoingreference is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the synthesis of 5-azacytidine (also known asazacitidine and 4-amino-1-β-D-ribofuranosyl-S-triazin-2(1H)-one).5-azacytidine may be used in the treatment of disease, including thetreatment of myelodysplastic syndromes (MDS).

BACKGROUND OF THE INVENTION

5-azacytidine (also known as azacitidine and4-amino-1-β-D-ribofuranosyl-S-triazin-2(1H)-one; Nation Service Centerdesignation NSC-102816; CAS Registry Number 320-67-2) has undergoneNCI-sponsored trials for the treatment of myelodysplastic syndromes(MDS). See Kornblith et al., J. Clin. Oncol. 20(10): 2441-2452 (2002)and Silverman et al., J. Clin. Oncol. 20(10): 2429-2440 (2002).5-azacytidine may be defined as having a formula of C₈H₁₂N₄O₅, amolecular weight of 244.20 and a structure of

The s-triazine ring of 5-azacytidine has a particular sensitivity towater (see J. A. Beisler, J. Med. Chem., 21, 204 (1978)); thischaracteristic has made the synthesis of 5-azacytidine a challenge,especially in manufacturing at commercial scale. A number of prior artmethods have been developed in order to avoid the use of water; however,these methods all have additional problems that render them undesirablefor the production of large-scale batches of 5-azacytidine. For example,Piskala and Sorm teach the following synthesis scheme in (see U.S. Pat.No. 3,350,388; A. Piskala and F. Sorm, Collect. Czech. Chem. Commun.,29, 2060 (1964); and A. Piskala and F. Sorm, Ger. 1922702 (1969), eachof which is incorporated herein by reference

in its entirety):

The overall yield of this scheme is 43.3%. This method involves areactive starting material (isocyanate) with a controlledstereochemistry (1-β configuration). Such a compound cannot be regardedas a starting material. The drawbacks of this scheme include thepresence of steps that are difficult to scale-up, the use of benzene assolvent in one step, and the requirement for a deprotection stepperformed in a closing pressure vessel using dry ammonia. Furthermore,the final 5-azacytidine product was isolated from the reaction mixtureby filtration with no further purification; this is not acceptable forthe synthesis of an Active Pharmaceutical Ingredient (API) for humanuse. The addition of further purification steps will further reduce theoverall yield.

Winkley and Robins teach an 5-azacytidine synthesis process that relieson the coupling of a “bromosugar” with a silyl derivative of5-azacytosine (see M. W. Winkley and R. K. Robins, J. Org. Chem., 35,491(1970), incorporated by reference in its entirety):

In this procedure, 5-azacytosine was treated with excesshexamethyldisilazane (HMDS) in the presence of catalytic amounts ofammonium sulfate at reflux until a complete solution was generated(TMS=(CH₃)₃Si). See E. Wittenburg, Z. Chem., 4, 303 (1964) for thegeneral procedure. The excess HMDS was removed by vacuum distillationand the residue was used directly (without further purification) in thecoupling with, 2,3,5-tri-O-acetyl-D-ribofuranosyl bromide inacetonitrile. The coupled product was deprotected with methanolicammonia solution.

There are many significant weaknesses in this procedure. First, the factthat the bromosugar was a mixture of anomers, which means that the finalcoupled product was also a mixture of anomers. Second, the work-up inthe coupling step involved a great many steps, specifically:concentration of the reaction mixture to dryness; treatment of theresidue with sodium bicarbonate, water and methanol; removal of thewater by co-evaporation with absolute ethanol; extraction of the residuewith chloroform twice; and finally the concentration to dryness of thecombined chloroform extract. Third, ammonia in MeOH was used in thedeprotection step, which requires the use of a pressure vessel. Fourth,the crude 5-azacytidine was isolated in only a 35% yield. This crudematerial was then dissolved in warm water and the solution wasdecolorized with charcoal. Evaporation then gave crystals of5-azacytidine with a yield 11%. This material was further recrystallizedfrom aqueous ethanol (charcoal). The low recovery during purificationcan be correlated with the poor anomeric ratio and with the known lowstability of 5-azacytidine in water.

Piskala and Sorm also teach the following process for coupling involvingthe use of a “chlorosugar” (A. Piskala and F. Sorm, Nuci. Acid Chem. 1,435 (1978), incorporated herein by reference in its entirety):

2,3,5-Tri-O-Benzoyl-D-ribofuranosyl chloride was prepared by saturatinga solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribose inCICH₂CH₂Cl—AcCl with gaseous HCl (with ice-cooling) and then keeping themixture overnight at room temperature. This procedure is difficult toscale-up with plant equipment due to the special handling requirementsof gaseous HCl. Also, the typical α/β ratio in the chlorosugar isunknown, as is the impact of the α/β ratio on the yield and final purityof 5-azacytidine.

Piskala, Fiedler and Sorm teach a procedure for the ribosylation ofsilver salts of 5-azapyrimidine nucleobases in A. Piskala, P. Fiedlerand F. Sorm, Nucleic Acid Res., Spec. Publ. 1, 17 (1975), incorporatedherein by reference in its entirety. Specifically, they teach that theribosylation of the silver salt of 5-azacytosine with2,4,5-tri-O-benzoyl-D-ribosyl chloride gives 5-azacytidine. This isclearly not a procedure that is amenable to scale up for the large-scaleproduction of 5-azacytidine.

Niedballa and Vorbrüggen teach the procedure that has been usedhistorically for the large-scale synthesis of 5-azacytidine for theabove-mentioned NCI-sponsored trials for the treatment ofmyelodysplastic syndromes. See H. Vorbrüggen and U. Niedballa, Ger.2,012,888 (1971) and U. Niedballa and H. Vorbrüggen, J. Org. Chem., 39,3672 (1974), each of which is incorporated herein by reference in itsentirety. The procedure involves the following steps:

There are at least three major drawbacks to this procedure. First, andmost importantly,

after purification, variable amounts of tin from one batch to anotherwere found in the API. The lack of control of the tin level means thatthe procedure is not suitable for producing an API for human use.Second, emulsions developed during the workup of the coupling mixture.Indeed, H. Vorbrüggen and C. Ruh-Pohlenz in Organic Reactions, Vol. 55,2000 (L. A. Paquette Ed., John Wiley & Sons, New York), p 100, havepreviously noted that silylated heterocycles and protected 1-O-acyl or1-O-alkyl sugars in the presence of Friedel-Crafts catalysts like SnCI₄often form emulsions and colloids during work-up. The phase separationof the emulsion is slow, so the water-sensitive protected 5-azacytidinewas exposed to water for variable periods of time leading to variableamounts of decomposition. Third, a filtration step was performed inorder to isolate the insoluble tin salt. Typically, this filtration isvery slow, and is likely the reason that variations in the final yieldwere noted. These problems mean that the process is not convenientlyamenable to scale-up.

Vorbrüggen et al. in Chemische Berichte, 114: 1234-1255 (1981) teach theuse of certain Lewis acids as Friedel-Crafts catalysts for the couplingof silylated bases with 1-O-acyl sugars. In particular, they teach thecoupling of silylated bases with 1-O-acyl sugars in the presence oftrimethylsilyl trifluoromethanesulfonate (TMS-Triflate) in1,2-dichloroethane or acetonitrile. The reaction mixture was thendiluted with dichloromethane and the organic phase extracted withice-cold saturated NaHCO₃. The use of this procedure to synthesize5-azacytidine is not taught or suggested.

Vorbrüggen and Bennua in Chemische Berichte, 114: 1279-1286 (1981) alsoteach a simplified version of this nucleoside synthesis method in whichbase silylation, generation of the Lewis acid Friedel-Crafts catalyst,and coupling of the silylated base to the 1-O-acyl sugar takes place ina one step/one pot procedure employing a polar solvent such asacetonitrile. Following reaction, dichloromethane is added, and themixture is extracted with aqeous NaHCO₃. The use of this procedure tosynthesize 5-azacytidine is not taught or suggested. Moreover, this onestep/one pot reaction is not suitable for the synthesis of 5-azacytidinebecause the extraction is done is the presence of acetonitrile.Acetonitrile is a polar solvent, and is therefore miscible with water.As a consequence, the protected 5-azacytidine in the acetonitrile isexposed during extraction to the aqueous phase for variable amounts oftime, which in turn leads to variable amounts of decomposition of theprotected 5-azacytidine.

Thus, there is an unmet need in the field for the provision of a simple,controlled procedure for the synthesis of 5-azacytidine that provides anAPI that is suitable for use in humans, minimizes the exposure of5-azacytidine to water, and is amenable to scaling-up for the productionof large quantities of 5-azacytidine.

SUMMARY OF THE INVENTION

The present invention provides for the first time a method thatsynthesizes 5-azacytidine that is suitable for use in humans and isamenable to large scale synthesis.

In one series of embodiments, 5-azacytidine is prepared by:

a) reacting 5-azacytosine with a silylating reagent to yield a compoundof the structure:

wherein each R₁ is an optionally substituted C₁-C₂₀ alkyl groupindependently selected from the group consisting of straight chain alkylgroups, branched alkyl groups, and cyclic alkyl groups;b) coupling (A) with a compound of the structure:

wherein each R₂ is an optionally substituted C₁-C₂₀ acyl groupindependently selected from the group consisting of straight chain acylgroups, branched acyl groups, and benzoyl groups, wherein the couplingof (A) and (B) is carried out in the presence of trimethylsilyltrifluoromethanesulfonate (TMS-Triflate), and wherein the couplingyields a compound of the structure

andc) removing said Si(R_(i))₃ and R2 groups from (C).

In preferred embodiments, the silylating reaction takes place in theabsence of a solvent using an excess of silylating reagent, andoptionally in the presence of a catalyst. If a catalyst is used, apreferred catalyst is ammonium sulfate. Preferably the silylatingreagent is a trimethylsilyl (TMS) reagent (i.e., R₁=CH₃), or a mixtureof two or more TMS reagents in excess over the 5-azacytosine. PreferredTMS reagents include hexamethyldisilizane (HINDS) andchlorotrimethylsilane (TMSCl). The silylated 5-azacytosine is preferablyisolated prior to coupling by removing the silylating reagents usingvacuum distillation, or by filtration.

Preferably, the compound (B) of coupling step b) is

wherein B_(z)=

and the coupling reaction is carried out in a dry organic solvent, morepreferably a dry organic non-polar solvent that is not miscible withwater. Most preferably, the TMS-Triflate is quenched by extracting thereaction product of b) with, for example, an aqueous bicarbonatesolution.In another series of embodiments, a “one pot” synthesis of 5-azacytidineis provided comprising the steps of:

a) in a dry organic solvent, reacting 5-azacytosine with one or moresilylating reagents to yield a compound having the structure;

wherein each R₁ is an optionally substituted C₁-C₂₀ alkyl groupindependently selected from the group consisting of straight chain alkylgroups, branched alkyl groups, and cyclic alkyl groups;

b) adding directly to the reaction mixture of a) TMS-Triflate and acompound having the structure

wherein each R₂ is an optionally substituted C₁-C₂₀ acyl groupindependently selected from the group consisting of straight chain acylgroups, branched acyl groups, and benzoyl group to yield a compoundhaving the structure;

c) extracting the reaction mixture of b) with an aqueous quenchingsolution; and

d) removing said Si(R_(i))₃ and R₂ groups.

Preferably, the dry organic solvent of step a) is a polar solvent, mostpreferably acetonitrile. Preferably the polar solvent is removed betweensteps b) and c) and the reaction products of b) are dissolved in a dryorganic non-polar solvent, most preferably dichloromethane of1,2-dichlorethane, prior to step c).

In some embodiments, the crude 5-azacytidine produced by theabove-described processes is subjected to one or more recrystallizationprocedures. For example, the crude 5-azacytidine may be dissolved indimethylsulfoxide (DMSO), and then recrystallized by the addition ofmethanol.

The methods provided by the instant invention are amenable to scale-up,and avoid the use of tin catalysts and other metal ions, therebyproviding 5-azacytidine that is suitable for use as an API. The methodsalso avoid the formation of emulsions during the work up(quenching/extraction) of the coupling reaction, thereby avoidinghydrolysis of the s-triazine ring.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the most basic embodiment of the invention, 5-azacytidine issynthesized according to the following process wherein each R₁ is anoptionally substituted C₁-C₂₀ alkyl group independently selected fromthe group consisting of straight chain alkyl groups, branched alkylgroups, and cyclic alkyl groups, and wherein each R2 is an optionallysubstituted C₁-C₂₀ acyl group independently selected from the groupconsisting of straight chain acyl groups, branched acyl groups, andbenzoyl (Bz) groups.

According to this scheme, 5-azacytosine (1) is reacted with a silylatingreagent to yield a silylated 5-azacytosine (2). Preferably, thesilylating reagent is a trimethylsilyl (TMS) reagent or a mixture of twoor more TMS reagents. Preferred TMS reagents includehexamethyldisilizane (HMDS: (CH₃)SiNHSi(CH₃)₃) and chlorotrimethylsilane(TMSCl: (CH₃)₃SiCl). The silylated 5azacytosine is then reacted with aprotected β-D-ribofuranose derivative (3) in the presence ofTMS-Triflate (trimethylsilyl trifluoromethanesulfonate). TMS-Triflatecatalyzes the coupling reaction, resulting in the formation of aprotected 5-azacytidine (4). The protecting groups can be removed by anytechnique known in the art, including, but not limited to, treatmentwith methanol/sodium methoxide. The individual reactions of the schemewill now be discussed in detail.

Preparation of Silylated 5-Azacytosine

In one embodiment, the silylated 5-azacytosine is prepared by heating asuspension of 5-azacytosine (1), one or more TMS reagents (present inexcess over the 5-azacytosine) and a catalyst, preferably ammoniumsulfate, at reflux without a solvent until a clear solution results.Most preferably, the TMS reagent is HMDS, which produces atrimethylsilyl 5-azacytosine derivative (R₁═CH₃ in the scheme above). Bycooling to ambient temperature, the silylated 5-azacytosine crystallizesfrom the reaction mixture. The silylated 5-azacytosine can then beisolated by any technique known in the art. For example, the silylated5-azacytosine may be isolated by partially removing excess TMS reagent,followed by addition of a suitable solvent (for example, heptane) andfiltration under inert atmosphere. The silylated 5-azacytosine thusisolated is used with or without drying in the coupling step.Alternatively, silylated 5-azacytosine may be isolated by removing TMSreagent by vacuum distillation and then dissolving the residue is indichloromethane, acetonitrile, or 1,2-dichloroethane for use in thecoupling step.

In another embodiment, the silylated 5-azacytosine is prepared “in situ”from 5-azacytosine and an equivalent amount of one or more silylatingreagents (preferably a mixture of HMDS and TMSCl) in a suitable solventin the presence or absence of a catalyst at reflux. Preferably, thesolvent is a dry organic solvent, more preferably a dry polar organicsolvent, including but not limited to acetonitrile. The resultingsilylated 5-azacytosine can be used directly in the coupling stepwithout isolation as described below.

Coupling of Silylated 5-Azacytosine to Sugar

In one embodiment of the invention, coupling of the silylated5-azacytosine to the sugar is performed by first preparing a cooledmixture (preferably in the range of about 0° C. to about 5° C.) ofsilylated 5-azacytosine and 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (or1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose) in dichloromethane,acetonitrile, or 1,2-dichloroethane. Preferably, the solvent for thecoupling step is dichloromethane or 1,2-dichloroethane, most preferablydichloromethane. TMS-triflate is then added to the mixture, preferablyat a rate that keeps the temperature below 25° C. After the addition iscomplete, the clear solution is stirred at ambient temperature for about2 hours to about 3 hours.

In embodiments which the silylated 5-azacytosine is generated “in situ,”the coupling reaction mixture may instead be prepared by adding thesugar and TMS-Triflate directly to the silylation reagents (silylatingagent and 5-azacytosine). The sugar and TMS-Triflate can be addedconcurrently with the silylating reagents, or they may be added at theconclusion of the silylation reaction. Preferably, the TMS-Triflate andthe sugar are in the same solvent as used in the silylation reaction,which solvent, as described above, is preferably a dry organic polarsolvent including, but not limited to acetonitrile. Using “in situ”generated silylated 5-azacytosine in this manner thus allows one toperform “one pot” silylation and coupling. See Examples 5 and 6.

In embodiments where acetonitrile or other polar solvent is presentduring the coupling reaction (for example, in embodiments where “onepot” silylation and coupling are performed in a polar solvent), theacetonitrile or other polar solvent is first removed, preferably invacuum, and the residue is dissolved in dichloromethane or1,2-dichloroethane prior to quenching. Because polar solvents such areacetonitrile are miscible with water, removing such solvents from thecoupling product and then dissolving the product in dry organicnon-polar solvents such as dichloromethane or 1,2-dichloromethaneminimizes the exposure of the water-sensitive 5-azacytidine to theaqueous phase during extraction/quenching.

Quenching/extraction preferably is performed in a 1/1 w/w NaHCO₃/Na₂CO₃solution at about 0° C. to about 5° C. Using cooled quenching solutionfurther minimizes the decomposition of the protected 5-azacytidineproduct during quenching. The organic phase of the quenched reaction isthen separated and the water phase extracted with dichloromethane or1,2-dichloroethane. The combined organic extract is washed with cooled(preferably in the range of about 0° C. to about 5° C.) NaHCO₃ solution(preferably 10%) and water, then dried over MgSO₄, filtered, and thefiltrate concentrated in vacuum. The residue is a protected5-azacytidine (4). Methanol is then charged to the residue. Whendichloromethane is used (either as the coupling solvent or following useof acetonitrile as the coupling solvent), the dichloromethane may bepartially removed in vacuum, followed by charging methanol to themixture, and finally by continued vacuum distillation was continueduntil substantially all dichloromethane is removed.

As described above, the exposure of protected 5-azacytidine to water canbe minimized by using a non-polar dry organic solvent for the couplingstep. Alternatively, if a dry organic polar solvent is present at thecoupling step, that solvent can be removed and replaced with a drynon-polar organic solvent prior to quenching. The duration of exposureof the protected azacitidine to water (during quenching) also depends onthe size of the batch that is processed as small batches can beprocessed in a shorter time than large batches. Thus, in preferredembodiments of the invention, a single batch of coupling reactionproduct is split into smaller sub-batches, and each sub-batch isseparately subjected to quenching/extraction.

In preferred embodiments, the protecting groups are removed from theprotected 5-azacytidine (4) by diluting the methanolic solution ofprotected 5-azacytidine (4) with methanol, then adding sodium methoxidein methanol (preferably about 25% w/w) to the mixture with stirring atambient temperature. During this procedure, a white solid separates. Themixture is preferably left stirring for about 8 hours to about 16 hours,following which the solid is filtered off and washed with methanol(until the filtrate is about pH 7). The solid is then dried, preferablyin vacuum at about 55° C. to about 65° C. until the weight of the solidremains constant. The solid is crude 5-azacytidine (5).

The crude 5-azacytidine (5) may be purified by any technique known inthe art. In preferred embodiments, purification is performed bydissolving the crude product in dimethyl sulfoxide (DMSO) at about 85°C. to about 90° C. under stirring and in an inert atmosphere. Methanolis gradually added to the resulting solution under slow heating, and themixture is stirred at ambient temperature for about 8 hours to about 16hours. The resulting recrystallized solid is filtered off, washed withmethanol, and then dried, preferably under vacuum at about 85° C. toabout 95° C. until the weight remains constant. The overall yield isabout 30-40%.

The 5-azacytidine synthesis methods provided by the invention provides anumber of clear advantages over the prior art methods. First, themethods allow the manufacturing of pilot plant scale uniform batches of5-azacytidine. Second, the procedure assures an API without tin or othermetallic ion contaminants. Third, there are no difficult to handle phaseseparation (emulsion) problems in the work-up of the coupling step.Fourth, by removing polar solvents from the coupling reaction prior toquenching/extraction and then dissolving the reaction product isdichloromethane or 1,2-dichloroethane, the exposure of thewater-sensitive 5-azacytidine to the aqueous phase is minimized.Finally, the decomposition of the water-sensitive 5-azacytidine isfurther minimized during the quenching/extraction step by using cooledquenching solutions.

The following examples are provided for illustrative purposes only. Theyare not to be interpreted as limiting the scope of the invention in anyway.

EXAMPLES

In a 22 L, 3-necked flask, a mixture of 5-azacytosine (1) (2.0 kg, 17.8mol, 1.07 molar eq.), HMDS (9.162 kg) and ammonium sulfate (40.0 g) washeated at reflux for 2 hours. A fresh amount of ammonium sulfate (20.0g) was added, and the reflux was continued for 6 hours longer. Theinitial slurry turned into a clear, pale-yellow, solution and no moregas evolved at the end of the reflux. The excess HMDS was evaporated offin vacuum to obtain an off-white residue, which is trimethylsilylated5-azacytosine (6).

Trimethylsilylated 5-azacytosine (6) prepared according to the method ofExample 1 was diluted with anhydrous dichloromethane (18.1 kg) in a 50L, 3-necked, flask and solid, 1,2,3,5-Tetra-O-acetyl-β-D-ribofuranose(5.330 kg, 16.7 mol) (7) was charged to the mixture. An anhydrousdichloromethane rinse (0.533 kg) was used and the slurry was cooled to0-5° C. TMS-triflate (4.75 kg, 1.2 molar eq.) was added to the mixtureover 5-10 minutes. During the addition, the reaction temperatureincreased to 15-20° C., and the initial suspension turned into a clear,pale-yellow, solution. After 2 hours of stirring, the solution waspoured over a mixture of Na₂CO₃ (2.00 kg), NaHCO₃ (2.00 kg), water (29.9kg) and ice (20.0 kg). The layers were separated. The water layer wasextracted with dichloromethane (8.0 kg). The combined organics werewashed with cold (0-5° C.) 10% NaHCO₃ (2×10 L). The combined washingswere extracted with dichloromethane (8.0 kg). The combined organics werewashed with cold water (2×5 kg), dried on MgSO₄ (2.0 kg), and filtered.The filtrate and dichloromethane washes on the pad (2×1.32 kg) werecombined and reduced in volume using vacuum (˜200 mmHg, 30° C.). Thedistillation was continued until the majority of dichloromethane (app.85-95% total) was removed. The residue was taken up in methanol (4.0 kg)and the remaining dichloromethane was removed to give a protected5-azacytidine (8) as an off-white to yellow foam.

Protected 5-azacytidine (8) from Example 2 was diluted with methanol(35.5 kg), then 25% NaOMe in methanol (439 g, 0.11 mol. eq.) wascharged. The initial clear solution became turbid and a solid started toprecipitate. The slurry was left under nitrogen overnight. The solidswere isolated and washed with methanol (7×2.4 kg). The solids were dried(˜28 inHg and ˜85° C.) to a constant weight to give crude 5-azacytidine(1.835 kg; 44.9%) (5).

Example 4 Purification of Crude 5-azacytidine

Crude 5-azacytidine was purified from DMSO/MeOH as follows: Crude5-azacytidine (1.829 kg) was dissolved in preheated DMSO (5.016 kg;87-90° C.) under nitrogen. The solution was diluted with methanol inportions at approximate 10-minute intervals (9×1.4 kg then 1×0.58 kg)while slowly cooling. After the addition, 45-55° C. was maintained for 1hour and then the mixture was left to cool to ambient temperatureovernight. The next day, the solids were isolated at ambienttemperature, washed with Me0H (6×0.83 kg), and dried in vacuum (˜30 inHgand ˜85° C.) to a constant weight to give 5-azacytidine (1.504 kg; 82.2%recovery).

Example 5 One Pot Synthesis of 5-azacytidine

A mixture of 5-azacytosine (5.0 g, 44.6 mol), HMDS (6.3 mL, 29.8 mol),and TMSCl (6 mL, 47.3 mmol) in acetonitrile (78 mL) was heated to refluxfor 20 hours under an inert atmosphere. TMS-triflate (9 mL, 50 mmol) and1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (14.2 g, 44.6 mmol were addeddirectly to the silylated 5-azacytosine in acetonitrile. The additionwas performed at ambient temperature and under an inert atmosphere. Thereaction mixture was maintained under stirring for 20 hours, then pouredover a pre-cooled (0-5° C.) sodium bicarbonate solution (10%, 500 mL).The resulting mixture was extracted with dichloromethane (3×75 mL). Thecombined organic extract was washed with cooled (0-5° C.) 10% sodiumbicarbonate (2×25 mL) and brine (2×25 mL), then dried over magnesiumsulfate (10.0 g), filtered, and the filtrate concentrated in vacuum todryness. The off-white foam dissolved in methanol (120 mL) was treatedwith a solution of 25% sodium methoxide in methanol (1.0 g, 4.62 mmol).Soon a white solid started to separate. The suspension was stirred atambient temperature for 15 hours, then the solid was filtered off,washed with methanol (3×5 mL) and anhydrous ether (2×5 mL), then driedin vacuum. The crude 5-azacytidine (4.5 g, 41.3%) was further purifiedfrom DMSO and methanol (for details see Example 4).

Example 6 One Pot Synthesis of 5-azacytidine

A mixture of 5-azacytosine, HMDS, and TMSCl in acetonitrile is heated toreflux for 20 hours under an inert atmosphere. TMS-triflate and1,2,3,5-tetra-O-acetyl-β-D-ribofuranose are then added directly to thesilylated 5-azacytosine in acetonitrile. The addition is performed atambient temperature and under an inert atmosphere. The reaction mixtureis maintained under stirring for 20 hours, then the acetonitrile isremoved under vacuum. The solids are then dissolved in dichloromethane,and the mixture is poured over a pre-cooled (0-5° C.) sodium bicarbonatesolution (10%). The resulting mixture is extracted with dichloromethane.The combined organic extract is washed with cooled (0-5° C.) 10% sodiumbicarbonate and brine, then dried over magnesium sulfate, filtered, andthe filtrate concentrated in vacuum to dryness. The off-white foam isdissolved in methanol and treated with a solution of 25% sodiummethoxide in methanol. The suspension is stirred at ambient temperaturefor 15 hours, then the solid is filtered off, washed with methanol andanhydrous ether, then dried in vacuum. The crude 5-azacytidine isfurther purified from DMSO and methanol (for details see Example 4).

1. A method of preparing 5-azacytidine comprising the steps of: a)reacting 5-azacytosine with at least one silylating reagent to yield asilylated 5-azacytosine having the structure:

wherein each R₁ is an optionally substituted C₁-C₂₀ alkyl groupindependently selected from the group consisting of straight chain alkylgroups, branched alkyl groups, and cyclic alkyl groups; b) isolating thesilylated 5-azacytosine by removing the silylating reagent using vacuumdistillation or by filtration; c) coupling the isolated silylated5-azacytosine with a compound of the structure:

wherein the coupling is carried out in the presence of trimethylsilyltrifluoromethanesulfonate (TMS-Triflate) in at least one dry organicsolvent; d) quenching the reaction mixture of Step c) with an aqueousquenching composition comprising a bicarbonate salt and extracting thequenched reaction mixture; e) replacing substantially all of the solventin the extract of Step d) with methanol; f) deprotecting the product inthe extract of Step d) with sodium methoxide in methanol to yield5-azacytidine; and g) recrystallizing the 5-azacytidine product of Stepf) from hot dimethylsulfoxide.
 2. The method of claim 1 wherein eachsaid silylating reagent is a trimethylsilyl (TMS) reagent.
 3. The methodof claim 1 wherein each said silylating reagent is selected from thegroup consisting of hexamethyldisilizane (HMDS) andchlorotrimethylsilane (TMSCl).
 4. The method of claim 3 wherein saidsilylating reagent is HMDS.
 5. The method of claim 3 wherein saidsilylating reagents are HMDS and TMSCl.
 6. The method of claim 1 whereinsaid silylation reaction in step a) is carried out in the presence ofammonium sulfate.
 7. The method of claim 1 wherein each said dry organicsolvent is selected from the group consisting of dichloromethane and1,2-dichloroethane.
 8. The method of claim 1 wherein Step g) comprises:i) dissolving the product from Step f) in dimethylsulfoxide; ii) addingmethanol to the solution of i); and iii) isolating the recrystallizedproduct.
 9. The method of claim 1 wherein R₁ is methyl.
 10. The methodof claim 1 wherein the silylated 5-azacytosine from Step a) is isolatedby removing the silylating reagent using vacuum distillation.
 11. Themethod of claim 1 wherein the silylated 5-azacytosine from Step a) isisolated by filtration.
 12. The method of claim 11 wherein thefiltration is performed in the presence of heptane.
 13. The method ofclaim 11 wherein the filtration is performed under inert atmosphere. 14.The method of claim 1 wherein the aqueous quenching composition of Stepd) comprises an aqueous solution of sodium carbonate and sodiumbicarbonate.
 15. The method of claim 14 wherein the sodium carbonate andsodium bicarbonate have a weight ratio of about 1:1.
 16. The method ofclaim 1 wherein the aqueous quenching composition of Step d) is cooledto a temperature of between about 0° C. and about 5° C.
 17. The methodof claim 1 wherein the reaction of Step c) is performed indichloromethane.
 18. The method of claim 17 wherein the quenchedreaction mixture of Step d) is extracted with dichloromethane.
 19. Themethod of claim 18 wherein the dichloromethane extract is washed withcooled sodium bicarbonate solution.
 20. The method of claim 18 whereinthe dichloromethane extract is dried over MgSO₄ and filtered.
 21. Themethod of claim 18 wherein Step e) comprises: 1) removing at least someof the dichloromethane using vacuum distillation; 2) adding methanol;and 3) continuing vacuum distillation until substantially all of thedichloromethane is removed.
 22. The method of claim 1 wherein thereaction of Step f) is performed at ambient temperature.
 23. The methodof claim 1 wherein the product of Step f) forms a solid and separatesfrom the reaction mixture.
 24. The method of claim 1 wherein the productof Step f) is isolated by filtration.
 25. The method of claim 1 whereinStep g) comprises: 1) dissolving the 5-azacytidine product of step f) inhot dimethylsulfoxide; and 2) adding methanol to the solution of 1) andcooling the mixture.
 26. The method of claim 25 wherein the5-azacytidine product of step f) is dissolved in dimethylsulfoxideheated to a temperature of about 85° C. to about 90° C.
 27. The methodof claim 25 wherein the mixture of Step 2) is cooled to ambienttemperature.